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Detection and Characterization of Small Molecule Interactions with Fibrillar Protein Aggregates using Microscale Thermophoresis Emily Fisher, Yanyan Zhao, Robert Richardson, Malgorzata Janik, Alexander K. Buell, Franklin I. Aigbirhio, and Gergely Toth ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00228 • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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ACS Chemical Neuroscience

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Detection and Characterization of Small Molecule

2

Interactions with Fibrillar Protein Aggregates Using

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Microscale Thermophoresis

4 5 6 7 8 9

Emily Fisher1,§, Yanyan Zhao1,§, Robert Richardson1, Malgorzata Janik3, Alexander K. Buell2,4, Franklin I. Aigbirhio1, Gergely Tóth1,3, 5*

1

Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre, Department

10

of Clinical Neurosciences, University of Cambridge, Cambridge CB2 0QQ, UK

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2

Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK

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3

MTA-TTK-NAP B - Drug Discovery Research Group – Neurodegenerative Diseases,

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Institute of Organic Chemistry, Research Center for Natural Sciences, Hungarian

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Academy of Sciences, Budapest, Hungary

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4

Present address: Institute of Physical Biology, University of Düsseldorf, Germany

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5

Cantabio Pharmaceuticals Inc., Sunnyvale, USA

17 18

§

19

*

Both authors contributed equally to this work

Corresponding author email: [email protected]

1 ACS Paragon Plus Environment


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Abstract

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Neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease share the

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pathological hallmark of fibrillar protein aggregates. The specific detection of these

4

protein aggregates by positron emission tomography (PET) in the patient brain can yield

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valuable information for diagnosis and disease progression. However, the identification

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of novel small compounds that bind fibrillar protein aggregates has been a challenge. In

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this study, microscale thermophoresis (MST) was applied to assess the binding affinity of

8

known small molecule ligands of α-synuclein fibrils, which were also tested in parallel in

9

a Thioflavin T fluorescence competition assay for further validation. In addition, a MST

10

assay was also developed for the detection of the interaction between a variety of small

11

molecules and tau fibrils. The results of this study demonstrate that MST is a powerful

12

and practical methodology to quantify interactions between small molecules and protein

13

fibrillar aggregates, which suggest that it can be applied for the identification and

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development of PET radioligands and potentially of therapeutic candidates for protein

15

misfolding diseases.

16 17

Key words: Microscale thermophoresis; α-synuclein fibrils; tau fibrils; protein

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aggregation; binding affinity; fluorescence competition assay


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Introduction

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Insoluble protein fibrils are the pathological hallmark of several neurodegenerative

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diseases such as Parkinson’s disease (PD), characterized by intra-neuronal deposits of α-

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synuclein (α-syn) fibrils, and Alzheimer’s disease (AD), characterised by intracellular tau

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and extracellular Aβ40/42 fibrils.1,2 Imaging fibrillar proteins in vivo by the biomedical

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technique of positron emission tomography (PET), involving the use of selective

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radiotracers,3 could enable earlier diagnosis and improved understanding of disease

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progression. In addition, they could significantly impact treatment strategies and enable

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better assessment of new potential treatments. However, a major challenge in the field

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has been to identify novel molecular entities that selectively bind to a specific type of

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protein fibril due to the technical limitations in accurately and practically determining the

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binding affinities between potential new radiotracers and β-sheet-rich fibrils. 4, 5

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Although a variety of studies have been reported that describe the effects of small

14

molecules on aggregation and misfunction of α-syn and tau in vitro and in vivo, only a

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limited number of studies exist that characterize in detail the specific and high affinity

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binding of compounds to these proteins in their fibrillar state6-9. Compounds that have

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been reported to bind to fibrillar α-syn include: PiB10,11, Thioflavin T (ThT) 10,12, SIL512,

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SIL2612 and SU431213. Several publications have reported on interactions between tau

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fibrils and small molecules such as thiazine red13,

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Thioflavin S13 and radioligands such [18F]T80716, 17 and [11C]PBB318,19. Among those

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reports, radioligand binding assays and fluorescent competition assays are most widely

22

used to characterise the interaction between small molecules and fibrillar proteins.20 A

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, lansoprazole8,


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, astemizole8,


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number of biophysical methodologies have been used to study interactions between

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protein fibrils and small molecules.21 However, most of these methodologies have

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limitations either in sensitivity, accuracy or in their throughput and practical applicability.

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Microscale Thermophoresis (MST) is a relatively recent technique for the study of

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biomolecular interactions22-25 in an aqueous environment in which no immobilization of

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either reaction partner is required. MST exploits the phenomenon of ‘thermophoresis’ –

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the directed movement of molecules along a microscopic temperature gradient.26 The rate

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and direction of movement is dependent upon the size, charge and solvation shell of the

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species under study25. Any changes in this environment has the potential to change in the

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thermophoretic movement of the species.27 This technique has been shown to be capable

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of distinguishing very small changes to protein surfaces that occur, for example, when a

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small molecule binds to the surface of a large fluorescently labelled protein.27 A recent

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study by one of the co-authors of the present work described the investigation of the

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thermophoretic behaviour of oligomeric and fibrillar aggregates of α-syn and described

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binding experiments with MST between α-syn aggregates and EGCG, a green tea

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flavonoid as well as a single chain camelid antibody.25

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This study aims to assess the general ability of MST to study the interactions between

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small molecules and protein fibrils for the discovery and development of novel small

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molecule PET radiotracers.


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Results

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Preparation of protein fibrils for MST experiments

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Amyloid fibrils of the proteins α-syn and tau were produced according to established

4

protocols (see Methods section).

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heterogeneous in nature after initial preparation (Figure 1). Both type of fibrils showed

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polydisperse length distributions and the tau fibrils also showed morphological diversity

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(straight vs. twisted). Such heterogeneity in fibril size and morphology may cause

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variations in signal readout when performing MST experiments, as distinct fibril

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morphologies may interact differently with small molecules and may cause

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inconsistencies in the quantities of fibrils pipetted into each. Further complication may

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arise from the fact that the thermophoresis of proteins can be size- and structure-

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dependent24. Therefore, it is important to homogenise the fibril sample before its

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application for MST experiments. α-Syn fibrils were sonicated in a water bath for 1

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minute prior to MST experiments in order to break up higher order assemblies of fibrils

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as well as to obtain a more uniform fibril size. This should be performed immediately

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prior to each experiment as some of our experiments suggested that sonicated fibrils

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display a difference in thermophoretic behaviour 2-3 hours after sonication (see Figure

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S1), potentially due to a partial dissociation at the high dilutions employed in MST

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experiments. Similarly, the homogenization of tau fibrils was attempted by sonication,

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however, unlike the α-syn fibrils, sonication was found to fragment the tau fibrils into

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very short pieces, which were subsequently observed to be unstable and not suitable for

Both α-syn and tau fibrils were observed to be



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MST experiments (Figure S2). Instead, magnetic stirring was used to homogenize tau

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fibrils just before the MST experiments.

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α-Syn fibrils were generated by mixing N122C cysteine variant protein monomers

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labelled with Alexa Fluor® 647 with unlabelled wild type monomers in specific ratios

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before growing the fibrils to generate fibrils with different fluorophore labelling

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densities24. In our studies labelling densities of 1 – 50 % were investigated, and it was

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found that 2 % labelled fibrils gave the most consistent results and showed no

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fluorescence quenching due to the presence of compounds. However, the use of 2 %

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labelled fibrils required a final concentration of 500 nM of α-syn fibrils to allow

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fluorescence readings within the recommended fluorescence range for MST

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measurements. Tau fibrils, on the other hand, were labelled through amide coupling,

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using the fluorescent probe NT647-NHS after the fibrils were grown (see Methods

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section for details). The fluorescence of the labelled tau fibrils was recorded, and the

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labelling ratio was calculated according to a calibration curve (Figure S3). The labelling

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ratio for the fluorescence probe on tau fibril was 0.8, which is within the typical range of

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labelling ratio for proteins with a stable tertiary structure of 0.5 to 1.1 according to the

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User Guide of MST.28 Tau fibrils were used at a concentration between 20 to 25 nM,

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which provided fluorescence values higher than the recommended 200 counts of our

19

instrument.

20 21

Detection and determination of the binding affinity of protein fibril – small molecule

22

interactions


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Fibrillar α-syn – small molecule interaction determination using MST

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The first compound investigated was SIL26, which has a reported affinity for fibrillar α-

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syn of Ki = 11.7-20.6 nM29 determined by radioligand competition assay with [123I]SIL23.

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During our experiments, various concentrations of SIL26 (highest concentration 100 µM)

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were incubated with α-syn fibrils (500 nM) and a combined (n = 3) binding curve was

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plotted as shown in Figure 2. From this, the Kd was determined to be 285.8 ± 191 nM.

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The second compound under assessment was SU4312 with a reported affinity of Ki = 490

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nM13 determined via a Thioflavin S based fluorescence assay. During our experiments,

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various concentrations of SU4312 (highest concentration 50 µM) were incubated with α-

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syn fibrils (500 nM) and a combined (n = 3) binding curve was plotted as shown in

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Figure 2. From this, the Kd was determined to be 457.3 ± 147.6 nM.

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The final compound assessed was ThT, which has been extensively studied in the

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literature for its affinity to protein fibrils. Reported affinities for α-syn range from Kd =

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588 ± 2 nM10 to 948 ± 271 nM12, both determined by ThT fluorescence saturation curves.

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The Kd has also been determined from a radioligand assay with [123I]SIL23 as Ki = 1040

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(755 – 1440) nM (95% CI)29. During our experiments, various concentrations of ThT

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(highest concentration 20 µM) were incubated with α-syn fibrils (500 nM) and a

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combined (n = 6) binding curve was plotted as shown in Figure 2. From this, the Kd was

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calculated as 3.10 ± 1.05 µM. It has been reported that in solution ThT forms micelles at

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concentrations above approx. 20 µM.30 Micellar ThT is likely to display a different mode

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of binding to fibrils compared to monomeric ThT, therefore concentrations above the

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reported critical micelle concentration were not investigated during these experiments. 7 ACS Paragon Plus Environment


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Fibrillar α-syn – small molecule interactions determined using a ThT fluorescence

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competition assay

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In order to validate MST as an appropriate biophysical method for determining fibril-

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small compound interactions, each of the compounds tested for its binding affinity to α-

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syn fibrils in this study was also assessed by the more established ThT fluorescence

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competition assay. Initially, we investigated the binding of ThT to unlabelled α-syn

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fibrils by titrating varying concentrations of ThT (30 – 0.03 µM) into a fixed

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concentration of α-syn fibrils (0.5 µM) and subsequent measurement of the resulting

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fluorescence intensity. From these experiments the (apparent) Kd was calculated as 3.13 ±

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0.76 µM (n = 8) (Figure S4).

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Next Ki values for SIL26 and SU4312 with α-syn fibrils were determined by a ThT

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fluorescence competition assay. We incubated a fixed fibril concentration (0.5 µM) with

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10 µM ThT and varying concentrations of SIL26 (1 nM - 300 µM) and SU4312 (0.5 nM

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– 150 µM). The underlying assumption of this assay is that ThT and the compound under

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investigation compete for the same binding sites on the fibrils, and that there is no direct

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interaction between ThT and the respective compound. It is clear that these assumptions

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are not always fulfilled, as we have shown before.31 The results obtained from these

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competition experiments, if interpreted in the framework of the assumptions mentioned

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above, were Ki = 4067 ± 1120 nM (n = 4) for SIL26, and Ki = 248 ± 65 (n = 5) for

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SU4312 (Figure S5 and S6 respectively).

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Fibrillar tau – small molecule interaction determination using MST


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Firstly, the interactions between tau fibrils and thiazine red and T807 were investigated at

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room temperature using MST. The affinity of thiazine red to tau fibrils was reported to be

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Ki = 200 nM13 determined by a Thioflavin S competition assay. The affinity of T807 to

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tau fibrils was reported to be Kd = 14.6, 15 nM16,17 determined via a radioligand binding

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assays. During the MST experiments, various concentrations of thiazine red and T807

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(highest concentration - 10 µM) were added to tau fibrils (50 nM), and from the binding

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curves (Figure 3) the Kd was determined to be 123 ± 47.4 nM (n = 3) for thiazine red and

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Kd = 39.7 ± 18.2 nM (n = 2) for T807. Interestingly, we observed significant variation in

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the resulting binding curves among the distinct set of experiments for thiazine red

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interacting with tau fibril. Shown in Figure 4, there are three groups of repeats of the

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thiazine red - tau fibril MST experiments. The first data set (triangles) shows a significant

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change in the thermophoretic behaviour of the tau fibrils due to an interaction with

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thiazine red. The second data set (squares) still shows a thermophoresis change but it is

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less pronounced than in the first set, while, the third data set (crosses) shows no sign of

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an interaction between thiazine red and tau fibrils.

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Next, MST experiments were carried out to detect the interaction between lansoprazole

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as well as a tau binding compound32, 582407, with tau fibrils. 582407 was identified in a

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high throughput biophysics based screen that detected the interaction between small

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molecules and monomeric tau protein.32 The binding affinity value of lansoprazole to tau

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fibrils was reported to be Ki = 2.5 nM8 determined by competition assay with

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radiolabelled compound 3H-astemizole and Kd = 3.3 nM8 by radioligand binding assays.

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During these MST experiments, various concentrations of lansoprazole (highest

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concentration 10 µM) and 582407 (highest concentration 12 µM) were added to tau 9 ACS Paragon Plus Environment


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fibrils (25 nM for lansoprazole and 20 nM for 582407) in separate experiments and

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binding curves (n = 2) were obtained (Figure 3). From these curves the Kd for

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lansoprazole was calculated as 33.8 ± 8.37 nM, and the Kd for 582047 was calculated as

4

2.02 ± 0.647 nM. Similarly to the thiazine red experiments, variation in the Kd values

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determined from these experiments was also observed (Figure S7).

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Background control experiments with Tau fibrils in 2% DMSO, 0.05% Tween 20,

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HEPES buffer and α-syn fibrils in 10% DMSO Tris buffer were also performed (shown

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in Figure S8). Insignificant deviations were observed compared to the binding transitions

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shown in Figures 2 and 3 suggesting that the MST measurements have a stable and

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repeatable background signal.

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Discussion

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In Table 1, we compared data from both the literature and fluorescence assay results with

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our results obtained from MST experiments. For SIL26, there is a significant difference

15

between the Ki obtained during our fluorescence assay experiment when compared to

16

literature result (Ki = 11.7-20.6 nM29), and the result obtained from our MST experiments

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(Kd = 285 ± 181 nM). This discrepancy might be caused by a failure of the competition

18

assays to accurately report on binding affinities in some cases, as discussed above. In

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particular, if ThT and SIL26 bind to different binding sites, the competition assay will

20

underestimate the affinity of SIL26.

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literature affinity data and the results obtained via MST for the ThT - α-syn fibril and

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lansoprazole - tau fibril systems. These differences are most likely due to differences in

Small differences can be observed between


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solution conditions used for growth of the protein fibrils. For example, in the case of α-

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syn the buffer used to grow the fibrils was 20 mM Tris-HCl, pH 8, 100 mM NaCl29

3

whereas herein, we used 20 mM phosphate buffer at pH 6.5. For the case of the tau fibrils

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according to literature experiments with lansoprazole, tau fibrils were grown using

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shaking or high salt concentrations (150 mM), whereas herein we did not apply shaking

6

and used a salt concentration of 5 mM. These differences in fibril growth conditions may

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cause differences in the fibril morphology, and hence may be responsible for the

8

differences in binding affinities. Furthermore, changes in solution conditions may modify

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the surface properties of the fibrils, and hence the binding affinity of the investigated

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compounds. In the case of SIL26, ThT and lansoprazole, we can conclude that given the

11

differences in experimental conditions and techniques employed in the different studies,

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the results from MST did not differ significantly from results reported in the literature.

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The same conclusion can made for the affinity of ThT obtained by fluoresence ThT

14

saturation binding study. For SU4312 binding to fibrillar α-syn and for thiazine red and

15

T807 binding with tau fibrils, good agreement was observed between the results obtained

16

by MST and those reported in the literature.

17

The variation in results obtained for separate sets of binding experiments to tau fibrils for

18

each compound could be partly due to the inherent variation observed within the

19

preparations of tau fibrils. Variations in the lengths, thicknesses and morphologies

20

(straight vs. twisted) of the tau filaments were observed in the case of heparin-induced

21

tau aggregation (Figure 1). The mass and morphological variations of tau fibrils in our

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experiments may lead to variations in the observed binding affinity of a compound to the

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tau fibrils as thermophoretic movement of protein aggregates has been found to be size11 ACS Paragon Plus Environment


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dependent24, and the relative proportions of individual morphologies may vary between

2

different batches of fibrils. Despite stirring in an attempt to improve homogeneity of the

3

tau fibrils, the intrinsically insoluble nature of fibrils as well as the small sample volume

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of the capillaries used in the MST experiments renders it challenging to maintain the

5

same concentration and morphological distribution of tau fibrils for all experiments.

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Sonication has been reported to be used for reducing the length heterogeneity of α-syn

7

fibrils.33, 34 However, the wild type (WT) tau fibrils used in this study were found to be

8

sensitive to sonication, i.e. that sonication appears to convert the fibrils into potentially

9

non-fibrillar species. It has been reported previously35, that sonication treatment is able to

10

disrupt the quaternary structure of WT tau fibrils, consistent with our results.

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The second reason for the variation of determined binding affinities could be due to the

12

low (20 - 25 nM) concentration of the tau fibrils used for the MST experiments. As all the

13

compounds tested here are reported or expected to have Kd values in the nM range, at

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such low concentration of tau fibrils the aggregates are thermodynamically unstable36, 37,

15

which means that the tau fibrils will dissolve over time. The kinetics of the dissociation

16

process depends on the temperature, solution conditions and the fibril length distribution.

17

Therefore, the exact concentrations of fibrils applied in the experiments were difficult to

18

estimate, and a fraction of the tau sample could be in monomeric and/or oligomeric form

19

rather than fibrillar, especially if the sample was allowed to evolve for several hours after

20

homogenisation. The variation of the different proportion of monomer, oligomer and

21

fibril within the measurement sample would result in different apparent Kd values, as we

22

have observed, especially as all these compounds have only been reported to bind to

23

fibrils. On the other hand, the use of very low concentrations of tau fibrils is necessary, 12 ACS Paragon Plus Environment

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because in order to determine the binding affinity of the compounds to tau fibrils, the

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concentration of fibrils should be near or slightly above the estimated Kd values.

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When comparing α-syn and tau MST data, it was observed that the binding curves

4

showed opposite S shapes, indicating a decrease in thermophoretic mobility upon small

5

molecule binding to tau fibrils and an increase in thermophoretic mobility for α-syn

6

fibrils in the case of the small molecules tested in this study. It is difficult to interpret

7

these observations, given the complexity of the phenomenon of thermophoresis and the

8

multitude of factors that influences the sign, and magnitude of the thermophoretic

9

mobility24.

10

It is important to discuss herein that the ThT fluorescence competition assay does not

11

allow the binding stoichiometry to be determined, because the quantity of bound ThT

12

cannot be determined from the fluorescence intensity alone. This should require a

13

physical separation between the bound and free ThT molecules, as can for example be

14

achieved using microfluidics.38 Furthermore, the Kd value determined through this

15

method corresponds to an apparent Kd because the emitted fluorescence intensity is not

16

ideally proportional to the quantity of bound ThT, especially at higher concentrations.

17

This is due to the so-called “inner filter effect”39, whereby due to the strong absorbance

18

of the ThT, not all parts of the solution experience the same excitation intensity (primary

19

effect), and also some fraction of the emitted fluorescence can be reabsorbed (secondary

20

effect). Moreover, more complex binding behaviour with multiple binding sites with

21

different affinities and stoichiometries, as has been reported for ThT binding to protein

22

fibrils40 , will be difficult to capture with this simple method. Nevertheless, this method



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allows rapid assessment of the order of magnitude of the binding affinity of ThT to a

2

given type of protein fibril under a given set of conditions.

3

In this study, the following two distinct fluorescence labeling strategies were applied to

4

protein fibrils: labeling of pre-formed fibrils, in the case of tau; and the formation of

5

fibrils from a defined proportion of unlabeled protein and protein that was labeled in a

6

defined position, in the case of α-syn. In the case of the later approach, fluorescent

7

labeling of the monomeric protein allows exquisite control over the proportion of labeled

8

subunits in the final aggregates. Furthermore, when the fluorescence label is placed at a

9

defined position in the monomer, which is possible when the protein is engineered to

10

have a cysteine residue, such as α-syn in our case in position 122. The disadvantage of

11

this strategy is that it has to be established in separate experiments that the labeling of the

12

monomer does not interfere with the aggregation process of the protein. This is the case

13

here, as we have established this in a previous study for the N122C mutant of α-syn41.

14

The non-specific fluorescence labeling of the fibrils does not allow this level of control,

15

but ensures that the fibrils are formed in an undisturbed manner. Furthermore, this

16

method does not necessitate the use of a specifically engineered monomer, and is hence

17

easier and more rapid. Because the aim of the present study is to establish MST as a

18

technique for detecting the binding of small molecule ligands to amyloid fibrils, and

19

therefore the presentation of both labeling strategies is an important part of the study.

20

In conclusion, our results show that MST can be a powerful, practical and rapid

21

methodology for quantifying interactions between small molecules and β-sheet-rich

22

fibrillar protein aggregates. The advantages of this methodology are due to the low

23

amounts of sample required for the experiments and due to the high sensitivity of the 14 ACS Paragon Plus Environment

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methodology. However, as highlighted here, there are some practical challenges for

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obtaining consistent results, primarily related to protein fibril sample preparations, which

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also should affect also binding experiments that use different biophysical methodology.

4

Therefore, MST is proposed to be an innovative methodology for identifying new

5

chemical entities that interact with β-sheet-rich fibrillar protein aggregates for developing

6

novel and specific PET radiotracer or therapeutic candidates targeting protein fibrils.

7 8

Materials and Methods

9

Preparation of α -syn and tau filaments

10

α-syn fibril preparation

11

Wild type and fluorescently labelled α-synuclein (α-syn) protein was produced as

12

reported previously.41 The labelled fibrils were prepared as per the procedure reported in

13

the recent publication by Wolff et al.25 as follows: 5% by mass of (unlabeled) wild-type

14

α-syn seed fibrils were incubated with a total of 50 µM of monomeric protein; 2% Alexa-

15

647 labelled N122C cysteine variant α-syn monomers and 98% unlabelled wild-type α-

16

syn monomers in 20 mM phosphate buffer pH 6.5. The samples were incubated at 37 °C

17

overnight, then they were diluted 1:2 into H2O, sonicated for 3 s and incubated at room

18

temperature overnight. Then the samples were flash frozen in liquid nitrogen and stored

19

at -80 °C until used.



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1

An equivalent protocol was followed for preparations of fibrils for fluorescence assay

2

experiments, using only unlabelled wild-type α-syn monomers and wild-type α-syn fibril

3

seeds.

4

Tau fibril preparation

5

Monomeric tau protein in the form of a lyophilized powder was obtained from Sigma-

6

Aldrich (Tau-441 human). Tau monomers were first dissolved in pure water (filtered with

7

a Milli-Q system) to give a 100 µM monomer solution in water. Buffer was then

8

exchanged to 20 mM BES buffer (pH 7.4 with 25 mM NaCl and 2 mM Dithiothreitol

9

(DTT)) using ultrafiltration filter (Amicon Ultra-0.5mL 10K centrifugal filter from

10

Millipore UK Ltd). The resulting solution was incubated at 56 °C for 10 min to destroy

11

the intramolecular disulfide bridge of compact monomer. After cooling to room

12

temperature in a water bath, heparin and protease-inhibitor-mix were added and then

13

incubated at 37 °C for 10 days, with addition of 2 mM fresh DTT each day.

14

Atomic Force Microscopy

15

Atomic force microscopy (AFM) images were taken using a Nanowizard II atomic force

16

microscope (JPK Instruments, Berlin, Germany) using tapping mode in air. α-syn AFM

17

images were acquired as per procedure reported in the recent paper published by M.

18

Wolff et al. 2016

19

water and 10 µl was deposited on freshly cleaved mica (Agar Scientific, Stansted, UK)

20

and left to dry. After 10 days of incubation, tau fibrils were diluted to 5 µM in HEPES

21

buffer and 10 µL was deposited on cleaned cleaved mica and left to dry. The sample then

25

. The samples were diluted to ∼1 µM total protein concentration in


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1

rinsed with water to remove any salt and then dried again. After a second rinse and dry

2

the sample was examined by AFM.

3

Fluorescence Spectroscopy

4

The formation of tau fibrils was also confirmed using Thioflavin S fluorescence.

5

Solutions composing 2 µM tau fibrils, 20 µM Thioflavin S, 10 mM HEPES buffer were

6

analysed at 440 nm (excitation) and 510 nm (emission) with an integration time of 1 s.

7

Measurements were record using Cary Eclipse Fluorimeter. Hellma® fluorescence

8

cuvettes (ultra Micro) were bought from Sigma-Aldrich.

9

ThT fluorescent competition assay

10

Measurements were made using Corning® 96 well plates, black polystyrene, NBSTM, non-

11

sterile. Fluorescence was measured using ‘Fluorescence Top Reading’ mode of Fluostar

12

Omega, BMG Labtech instrument fitted with an excitation filter of 450 nm and an

13

emission filter of 485 nm.

14

ThT Saturation Binding: For determining ThT Kd via saturation binding curve the

15

following was added to the plate; 40 µL Tris buffer (5.5 mM pH 7.4) spiked with 20%

16

DMSO, 20 µL of ThT solution (of varying concentrations between 30 µM and 0.03 µM

17

in Tris buffer) and 20 µL α-syn fibrils (2 µM in Tris buffer). The data was analysed using

18

non-linear fitting.

19

Determining Ki of SIL26 and SU4312: To determine an IC50 value for each compound, a

20

dilution series of concentration range 300 µM – 1 nM (SIL26) and 150 µM – 0.5 nM 17 ACS Paragon Plus Environment


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1

(SU4312) were prepared maintaining 20% DMSO throughout. 40 µL of these dilutions

2

were added to the plate alongside 20 µL ThT solution (40 µM in Tris buffer), 20 µL α-

3

syn solution (2 µM in Tris buffer). The data was analysed using non-linear fitting. The

4

IC50 value was used to calculate the Ki using the following equation, where Kd is the

5

apparent binding affinity between ThT and α-syn fibrils and was fixed at 3.129 µM:

=

+

[ ]

6

Microscale Thermophoresis

7

α-syn and Tau fibril labelling

8

α-syn fibrils were labelled with Alexa-647 with a labelling density of 2%. A small

9

portion of the stock solution was diluted to 1 µM with buffer and sonicated in a water-

10

bath (Branson 1510) for 1 min immediately prior to use.

11

10 µL, 100 µM tau fibrils were diluted to 200 µL with 10 mM HEPES buffer and then

12

added to a Slide-A-Lyzer Dialysis Cassette (20K MWCO, Fisher Scientific UK Ltd) to

13

remove the DTT in the fibril solution in order to avoid interference with the labelling

14

reaction. 42 After removing the DTT, 200 µL, 5 µM tau fibrils were mixed with 200 µL,

15

10 µM NT647-NHS dye and then incubated in dark for 30 minutes. Further dialysis (18

16

hours, changing the buffer after the first 4 hours) was used to remove the free dye giving

17

a 2.5 µM solution of labelled protein. The labelling ratio of dye unit per tau monomer

18

was then tested by a method recommended by the instrument manufacturer, NanoTemper


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1

Technologies, which is based on a calibration curve. The 2.5 µM tau fibril solution was

2

first aliquoted and then diluted to 10-50 nM just prior to use. The unused samples were

3

frozen at -20 °C.

4

MST measurement

5

α-syn: Experiments were performed using 1:1 mixture of α-syn fibril solution and

6

compound solution dilution series in standard grade capillaries (Nanotemper

7

Technologies, Munich, Germany). Samples were incubated at 25 °C within the capillaries

8

for 30 mins prior to running measurements. All measurements were conducted using

9

Monolith NT.115 instrument (NanoTemper Technologies, Munich, Germany) at 25 °C.

10

Assays were conducted at 70 % IR-laser Power and MST Powers (corresponding to the

11

excitation strength of the LED) of 40 % (SIL26, SU4312) or 80 % (ThT). The buffer

12

used for these experiments was 5.5 mM Tris, pH 7.4. Compounds SIL26, SU4312 and

13

ThT were initially dissolved in DMSO to afford stock solutions of 10 mM. These were

14

then diluted into buffer to initial concentrations of 100 µM (SIL26), 50 µM (SU4312)

15

and 20 µM (ThT) ensuring the final concentration of DMSO was 10%. Lower

16

concentration of SU4312 was required due to decreased solubility. During the MST

17

experiments, these compounds were serially diluted 1:1 with buffer containing 10%

18

DMSO to ensure a constant DMSO concentration throughout the dilution series.

19

Tau: Thiazine red, T807, lansoprazole and compound 582407 from

Detection and Characterization of Small Molecule ... - ACS Publications

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